This invention relates compositions and methods for making ungulate embryonic stem cells, producing chimeric ungulates from the stem cells, and deriving transgenic ungulates from the chimeras.
Although transgenic animals have been produced by several different methods in several different species, methods to readily and reproducibly produce transgenic large mammals such as ungulates at reasonable costs are still lacking.
Current methods for producing transgenic large animals such as ungulates, notably of the Order Artiodocylia, that includes, pigs, cattle, sheep and goats, have limitations that prevent them from becoming widespread in the commercial arena. Such methods include microinjection of ova and embryonic transduction with a recombinant molecule, for example, via a retroviral vector which includes a transgene.
To illustrate the high costs of such methods, microinjecting swine ova with genetic material to produce transgenic swine, costs between $25,000 to $250,000 to produce a single transgenic animal line. Another problem of microinjection is that it is a technically difficult procedure with an unacceptably low success rate. Furthermore, DNA transferred by microinjection is incorporated at random in the genome, usually in tandem linear arrays of multiple copies of the transgene. These limitations have resulted in animals being produced in which 1) the transgene is not incorporated at all, 2) the transgene is incorporated but not expressed, 3) the transgene is incorporated but expressed transiently or aberrantly. Rarely is the transgene incorporated and expressed normally. Also, the incorporation of transgenes in a host genome may result in the disruption of an endogenous gene by a so-called insertional mutation, which disrupts some aspect of the host's development, growth or normal physiology. Furthermore, random insertion results in difficulties controlling how the transgene will be regulated because flanking sequences upstream and downstream of the inserted transgenic DNA construct which can alter the control of the transgene expression are randomly associated with the transgene.
A method to generate transgenic animals, the use of transformed embryonic stem cells (ES-cells), has shown certain advantages over other methods when used to produce mouse chimeras, from which transgenic mice are derived. After they are isolated, ES-cells may be grown in vitro for many generations, producing unlimited numbers of identical ES-cells. These cells, when combined by fusion or injection with an early embryo, are capable of becoming part of the embryo and participating in the normal developmental process. The resultant animal is a chimera composed of two genotypes (Bradley et al., 1984).
An advantage of ES-cells is that they can be genetically manipulated in vitro. ES-cells may be transformed by introducing exogenous DNA into the ES host cells via electroporation or a biolistic approach. Following transformation, individual ES-cell clones may be screened in vitro for the incorporation and proper expression of the exogenous DNA before being used to produce chimeric embryos (Thomas et al., 1987).
The genetic manipulation of ES-cells in culture and the subsequent generation of transgenic animals via intermediate chimeric animals derived from the genetically manipulated ES-cells provide a particularly important advantage of ES-cell technology. Gene knockouts and gene replacements are methods of genetic manipulation via homologous recombination that have been carried out in microorganisms, but have only been practiced in mammalian cells within the past decade. These techniques allow for the targeted inactivation (knockout) of a particular gene, as well as for the replacement of a particular gene with an altered version of the gene, or with another gene. Such knockouts and replacements allow for alterations in the properties of cells and animals that cannot be readily achieved in any other way. The practice of mammalian gene knockouts and gene replacements, including the design of nucleic acid molecules and the detection of successfully altered mammalian cells is discussed in numerous publications, including Thomas et al., 1987; Jasin and Berg, 1988; Mansour et al., 1988; Brinster et al., 1989; Capecchi, 1989; Frohman and Martin, 1989; Hasty et al., 1991; Jeannotte et al., 1991; Mortensen et al., 1992; and Thomas et al., 1992. Therefore, both genetically novel and useful chimeric and transgenic animals may be produced.
Gene knockouts and gene replacements can be achieved through microinjection of mammalian zygotes. However, the number of zygotes that must be injected to practice these methods in this way are so high and the injections are so technically demanding as to render this approach extremely difficult and only one report of its successful accomplishment has ever been published (Brinster et al., 1989). Large enough numbers of ES-cells can be grown in culture and conveniently genetically manipulated by recombinant techniques in vitro to allow the routine production of gene knockout and/or gene replacements in the ES-cells and thereby in animals derived from the ES-cells.
ES-cell clones containing the transferred DNA can be selected and used for blastocyst injection. The ability to screen and select transformed ES-cells in vitro is one of the most important reasons for utilizing this strategy to produce transgenic animals. Use of whole animals proceeds only after it is known that the desired transformation was successful. This procedure minimizes in vivo failures, which are more expensive than in vitro tests and take longer to produce results.
A chimeric organism is one that is a mixture of cells which differ in their genetic complements. When transformed ES-cells are used to make chimeric embryos, some of these cells may be incorporated into the gonads of the chimera and participate in the formation of sperm and ova. Incorporation of the transgene into a gamete permits germ line transmission. Consequently, some of the descendants produced by chimeric individuals will be transgenic (Gossler et al., 1986; Robertson, 1987). A transgenic animal has the transgene in all of its cells, although the transgene is not necessarily expressed. It is not usually the individual that develops from the chimeric embryo that is transgenic, but rather offspring of that individual. This is an important distinction in as much as the chimeric individual can act as founder stock to produce many transgenic individuals that carry the desirable gene(s), but the chimera is not transgenic. However, the chimera is useful for the recovery of, new or heterologous genetic expression products, organs and the like.
ES-cells have been used to produce transgenic lines of mice that through homologous recombination have genes inserted into their genome at pre-selected sites. The strategy of creating animals with specific genomic changes has immense potential for genetic engineering in developing commercially valuable plants and animals, and in furthering understanding of the genetic control of mammalian development. However, the ES-cell method has not been successfully applied to production of larger transgenic mammals, for example, transgenic ungulates. A likely reason for the failure to extrapolate methods from mice to larger animals is the difference in developmental stages of the species. For example, the embryonic disc is not a solid mass in swine as it is in a 5-day old mouse.
Piedrahita et al. (1990a and b) isolated potential swine stem cells, but were unable to maintain lines or to demonstrate these cells' pluripotentiality. Pluripotent cells are defined as cells that are capable of being induced to develop into several different cell types. True totipotent embryonic cells are those capable of being induced to develop into any cell type present in an entire animal, that is, they have the potential to directly produce an entire animal. Ovine embryos did not produce ES-like cells at all. Porcine cell culture doubling time was 80 hours which is long relative to that of mouse ES-cells. The authors believed their presumptive porcine ES-cells were different from mouse ES-cells in morphology and behavior.
Notarianni et al. (1990) reported methods to produce transgenic pigs by use of pluripotent stem cells, but did not convincingly show that pluripotent embryonic stem cells were produced. Chimeric pigs were not reported as an intermediate step toward the production of a transgenic pig.
In International Publication No. WO90/03432, (hereinafter the “Evans” patent) and other publications from the Evans group, the conclusion was that “methods . . . for the isolation of embryonic stem cells from mouse embryos and successfully applied to hamster embryos are NOT applicable to ungulate embryos . . . ” (page 6) referring in particular to identification and isolation of stem cells, and predicted that ungulate “stem cells . . . would not necessarily resemble mouse embryonic stem cells in morphology or growth characteristics.” (page 7) The morphological description and figures illustrating some of the pig “selected cells” group, are more reminiscent of epithelial cells, than of embryonic stem cells from other organisms such as the mouse. Indeed, the authors state the “ES” cells from pigs are morphologically dissimilar from mouse ES-cells. Also, no biochemical tests were done to confirm that the selected cells were not differentiated. Chimeric animals were not shown as evidence that cells could differentiate into several cell types (pluripotency).
Even if some embryonic stem cells were actually mixed into the “selected” cell population reported by Evans, use of these cell populations to produce chimeric pigs would be expected to be relatively inefficient because chance would dictate whether an embryonic stem cell would be included in the material transferred to a host embryo. The probability of inclusion would be expected to be proportional to the percentage of embryonic stem cells in the mixed culture. The lack of a culture substantially enriched for ES-cells would lead to inefficient and unpredictable results. Moreover, the method disclosed could not be described as “a method to produce embryonic stem cells,” which implies substantial homogeneity and reproducibility, neither of which were demonstrated.
Evans teaches that a feeder cell layer is necessary for cell growth, and teaches away from the use of conditioned medium or growth factors. A feeder layer and the use of conditioned media were part of the methods of Piedrahita et al. (1990a and b) and Gossler (1986).
Strojek (1990) describes methods and results similar to those of Evans. Trophoblastic cells and non-homogeneous cultures derived from swine embryos were disclosed.
Handyside (1987) attempted to produce chimeric sheep from embryonic stem cells, but was admittedly unsuccessful. Flake (1986) produced chimeras from sheep, but resorted to in utero transplants rather than ES transfer.
Doetschman et al. (1988) identified “embryonic stem cells” from hamsters by growing them on mouse embryonic fibroblast feeder layers. Pluripotency was determined by differentiation in suspension cultures.
Ware (1988) reported embryo derived cells from “farm animals” growing on Buffalo Rat Liver (BRL) and mouse primary fetal fibroblasts.
Wall et al. (1991) suggested using transgenic swine as factories to produce biological products, but did not teach how to accomplish this goal.
Attempts to use embryonic carcinoma cells to produce chimeric mice by introducing such cells into an embryo, have had only limited success. Embryonal carcinoma cells were originally derived from embryonic cell tumors or teratocarcinomas (Stevens, 1970). Rossant and Papaioannou (1984) showed that both ES and ES-cells may differentiate in vitro into similar types. However, the formation of chimeric embryos exhibiting phenotypically normal development using ES-cells is usually low (Papaioannou et al., 1979; Rossant and McBurney, 1982), whereas ES cells are more efficient at producing chimeric mice (Bradley et al., 1984). In a variation on these methods, Martin (1981) reported growing mouse stem cells in media conditioned by the growth of teratocarcinoma cells. However, employing cancer cells in a growth environment is not likely to be palatable to the general public if such transgenic animals are ultimately to be used for products for human use, for example, food, or organs for transplants.
Improved methods for the production of chimeric and transgenic ungulates are clearly needed. A simple and efficient method is desirable to reduce costs and improve throughput. Chimeric and transgenic animals are useful as models for diseases for the testing of pharmacological agents prior to clinical trials or the testing of therapeutic modalities. Another advantage is that more desirable qualities in farm animals may be produced by introducing transgenes with suitable expression products to improve qualities. These desirable qualities include increased efficiency in feed utilization, improved meat quality, increased pest and disease resistance, and increased fertility.
Chimeric and transgenic animals are an alternative “factory” for making useful proteins by recombinant genetic techniques. Large animals such as pigs, cattle, sheep, and goats are potential factories for some products not obtainable from recombinant hosts such as microorganisms or small animals. Examples of such products are organs which are transplantable into humans.
Embryonic stem cell transfer to produce transgenic animals would be an improvement over available methods. A reason that embryonic stem cell-mediated gene transfer has not been employed in domestic livestock is the lack of established, stable embryonic stem cell lines available from these species. The availability of ES-cell lines would provide feasible methods to produce transgenic animals. However, development of ES-cell lines from livestock species is an extremely difficult process.
The early developmental embryonic morphologies of rodents (including mice) and ungulates (including swine) are quite distinct, particularly at the blastocyst stage. For example, the rodent blastocyst forms an egg cylinder, a tubular structure, while the ungulate blastocyst forms a developmentally equivalent flattened embryonic disk. The differences in the shapes of these otherwise equivalent structures contributes to the very different properties exhibited by the cells of rodent and ungulate blastocysts. These differences are most evident in vivo during the massive reorganization of cellular distribution that characterizes gastrulation in all animals. The migration and shape changes that the embryonic cells of rodents and ungulates must undergo during early development, and particularly during blastulation and gastrulation, are thus very different.
Differences in the properties of rodent and ungulate embryonic cells are also believed to be associated with the differences in placentation in these two groups of animals. Rodent trophoblast cells (the cells of the blastocyst that later form the placenta) are invasive in vivo and in vitro, while ungulate trophoblast cells are not invasive, and thus behave differently from rodent trophoblasts when cultured in vitro. With regard to the present invention, it is especially significant that in parallel with their different properties in vivo, many of the equivalent cells of rodent and ungulate embryos display different properties in vitro, as described above for trophoblast cells.
Thus, while the early embryonic development of all ungulates is morphologically identical, the differences between ungulate and rodent embryonic cells in vivo and in vitro have led to uncertainty in the art regarding the appropriate morphologic criteria to be applied to guide 1) the choice of ungulate embryonic cells to be isolated from embryos; 2) the selection of cells to be picked for expansion into useful ungulate ES-cell cultures; and 3) the selection of useful ungulate ES-cell cultures for continued propagation. These differences between ungulate and rodent embryonic cells in vivo and in vitro account for the significant differences in the practice (in accordance with the present invention) of embryonic stem cell isolation in ungulates, compared to the practice (as known in the art) of embryonic stem cell isolation in rodents.
Another problem in extrapolating from mice to ungulates, such as swine, is that exactly analogous developmental stages do not exist in the embryos of mice compared to ungulates. In ungulates, growth is generally slower, and the early embryonic ectoderm is present in a discoid arrangement, not as a solid mass as in the 5-day old mouse embryo.
In the present invention, limitations of the art are overcome by the production of stable, pluripotent ungulate embryonic stem cell cultures. These cell cultures are used to make chimeric ungulates, an intermediate step in producing a transgenic ungulate. Aspects of the invention differ from the art in, for example, culture conditions, validation of potency, and production of a chimera.
Several methods have been reported for the validation of ES cell lines from mammals. Studies in mice and Syrian Golden Hamsters have used cell morphology, biochemical markers, the ability to differentiate into various cell and tissue types and participation in embryonic development as criteria for validating the stem cell nature of embryonic cell lines. In swine, there have been several reports of the isolation of embryonic cell lines and inner cell mass cells from blastocysts. The criteria used for validation of an undifferentiated ES cell phenotype in these studies have included morphology, ability of the cells to differentiate in vitro into various cell and tissue types, and limited embryonic cell analysis of biochemical markers via immunocytochemistry and enzyme assays. However, none of the reported cell lines have produced chimeric offspring after the reintroduction of these cells into pre-implantation porcine embryos. A preferable validation for ES cells is their participation in embryonic development resulting in live chimeric offspring. The present invention is the first confirmed production of chimeric swine from ES cells.